Our long term goal is to learn how the self-renewal of stem cells is achieved and regulated, a question crucial to understanding tissue development, oncogenesis, wound repair, immunodeficiency, and infertility. Our focus is to study stem cells in the germline. In so doing, we hope to link our research between two extremely important yet highly enigmatic areas of biomedical research - germline and stem cell biology. Our approach is to establish Drosophila as a pilot system to explore molecular mechanisms underlying stem cell division, and to expand what we learn from Drosophila to mammalian systems via the reverse genetics approach. Specifically, we have identified germline stem cells in Drosophila and discovered key genes that control the intra- and intercellular mechanisms of the stem cell division. Further more, we have been intensively studying these genes in mammalian systems. This strategy has offered us unparalleled opportunities to discover fundamental stem cell mechanisms and to explore their medical significance. Our hypothesis is that the function of key stem cell genes is conserved during evolution. In support of this, we have discovered the first and only known family of evolutionarily conserved genes, the piwi family genes that are involved in stem cell self-renewal in diverse organisms in animal and plant kingdoms. Our working hypothesis in this proposal is that two murine members of this gene family, miwi and mili, are involved in cell-autonomous mechanisms that control spermatogonial stem cell division and spermatogenesis. Our specific aim in this proposal is to explore the stem cell mechanism by systematically characterizing the function of miwi and miii in spermatogonial stem cell division and its related processes. With all the tools in place and experience in most of the proposed analyses, we plan to: 1. Characterize mili expression during germline development. 2. Analyze the developmental function of miii by creating and examining conditional mili-null mice. 3. Characterize the roles of miwi and mili in spermatogonial stem cell division. 4. Analyze the biochemical roles of MIWI and MILl in spermatogonial stem cell division. 5. Identify direct target genes of MIWI and MILl to further study MIWI/MILI-mediated mechanisms.